The primary aim is to transfer a biotechnology platform for plant drug discovery from the academic laboratory to industry use (i.e. this application is directly responsive to the "lab to marketplace" roadmap program). Many "defensive" metabolites in plants are targeted on key proteins in the insect CNS, and homology between these and human proteins, makes such metabolites potentially valuable as CNS drugs. These plant metabolites evolved in response to natural selection, and so an imposed selection pressure should direct plant secondary metabolism toward more effective drugs. One way this could be achieved is by the expression of a human target protein, linked to a toxic mechanism, in plant cells. Now, cells which are producing inhibitors of this protein are more likely to survive, and repeated mutagenesis and selection should direct "evolution" toward increasingly effective inhibitors. For example, the human dopamine transporter (hDAT) is a molecular target in drug dependence and neurodegenerative disease. Expression of the functional hDAT in plant cells (Preliminary studies), makes these susceptible to neurotoxins, (MPTP, 6hydroxydopamine) which are transported by the hDAT. Conversely, the selective DAT inhibitor, GBR12909, protects these cells. Therefore in a mutant population, transgenic hDAT plant cells that survive these toxins should be "enriched" in clones which over-produce metabolites that inhibit the hDAT, or protect against the neurotoxins in some other way. Both would be of potential therapeutic value. To identify a species for this approach, extracts from ~1000 native plants were screened for relevant activity. This identified Lobelia cardinalis as the best candidate, and this species is also easy to culture and transform, making it ideal for proof of application. A population of transgenic hDAT L. cardinalis hairy roots, mutagenized by activation tagging, has now been subjected to MPTP selection. The proposal for phase 1 is to screen the resistant mutant sub-population for the presence of metabolites which inhibit the DAT in rat brain synaptosomes, or protect a human dopaminergic cell line against neurotoxins. Positive clones (phase 1 deliverables) will be used in phase 2 for compound identification, assessment of potential therapeutic value in complex models, and for regeneration into mutant medicinal plant lines. All of these products have potential commercial value, but it is the illustration of the general value of the technology, and its transfer from lab to marketplace, which is of most commercial value to the company. PUBLIC HEALTH RELEVANCE: Plants remain a very valuable source of complex bioactive compounds, but current methods of plant drug discovery do not compete with the pharmaceutical industry techniques of combinatorial chemistry and high throughput pharmacological screening. This proposal uses a novel biotechnology approach to the directed evolution and discovery of plant metabolites with potential value in human CNS diseases. These are among the most devastating and economically important diseases in the USA and world wide. The proposal focuses on potential medications for drug dependence and alcoholism, as well as for neurodegenerative conditions including psychostimulant-induced neurotoxicity, and diseases associated with aging, including parkinsonism and Parkinson's Disease (the second most common neurodegenerative disease in the US). The technology to be used combines plant molecular biology and molecular pharmacology, and its support will accelerate the introduction of this academic research into the commercial sector. Its simplicity and rapidity suggests that it may compete with the existing synthetic chemistry approaches, and might even revitalize the use of plants as a source of medicines for CNS disease.